Pluripotent stem cells have potential to differentiate into any of the three germ layers: endoderm, mesoderm, or ectoderm and provide a chance to obtain a renewable source of healthy cells and tissues to treat a wide array of diseases.
Methods currently used to detect/isolate pluripotent cells have inherent experimental variability and low efficiency, and are (1) mechanical isolation based on morphology that requires experience, and is laborious and not efficient; (2) quantification of the endogenous expression of stem cell transcription factors (OCT4, SOX2, etc.) in live cells, which requires genome modification; (3) fluorescence-activated cell sorting (FACS)-based analysis using cell surface markers (SSEA-4, TRA-1-60, etc.), which requires use of antibody based staining that is inherently variable; and (4) more recently, a pluripotent stem cell-specific adhesion signature, which is dependent on the surface properties of cell clusters and thus interrogates the population and not individual cells. Additionally, the identification of high-grade pluripotent hiPSCs is time consuming, requiring the generation of teratomas and several additional pluripotency test.
Several studies of chromosome territory occupation and genome distribution inside the nucleus show that the epigenome is dynamic and, that among other processes; it contributes to gene expression and cell differentiation.
Recent studies have revealed key differences in chromatin states of pluripotent cells as compared to differentiated cell types.
The spatial organization of chromatin inside the nucleus plays a key functional role. However, how nucleosomes are arranged to form the chromatin fiber is still highly debated.
The existence of a hierarchical organization of the chromatin fiber inside intact eukaryotic nuclei in vivo has recently been debated after cryo-electron microscopy, small-angle X-ray scattering (SAXS) and electron spectroscopic imaging experiments failed to detect the 30-nm fiber. The structural information obtained in these studies led to the overall conclusion that the eukaryotic nuclei are mainly composed of 10 nm fibers even though the core histone proteins could not be identified unequivocally using these methods due to their lack of molecular specificity. In addition, genome-wide analyses have revealed that nucleosomes are depleted at promoter and terminator regions and at many enhancers and that nucleosomes occupy preferred positions in genes and non-gene regions. Since the 30-nm fiber arrangement imposes specific constrains on nucleosome occupancy and positioning, these genome-wide analyses along with the latest imaging results argue against a hierarchical organization of nucleosomes along the chromatin fiber.
Conventional microscopy have shown that heterochromatin appears in large regions in pluripotent cells but it was confined to small foci in differentiated cells, confirming that chromatin in pluripotent cells assumes a globally more open conformation (Meshorer E. et al., 2006).
Up to date, however, the super-resolution studies of DNA and histones have not addressed questions regarding the organization of single or groups of nucleosomes, the overall nucleosome occupancy level of DNA and whether these parameters are consistent with the 30 nm fiber model of chromatin. How the chromatin organization changes at the nanoscale level as a function of cell state such as pluripotency and differentiation, while of fundamental importance, has also not been studied. In general, what has been lacking is a quantitative approach that can count and determine the number of nucleosomes within the chromatin fiber and thus identify nucleosome spatial arrangement at the nanoscale level.
Given the current debate on nucleosome occupancy, positioning and organization, and the importance of these parameters for DNA accessibility and gene expression, novel methods that allow quantitative visualization of nucleosome organization with high molecular specificity at the nanometer length scales in individual intact nuclei and leading to determine the chromatin state of a cell without the disadvantages of harsh sample preparation, lack of molecular specificity or low spatial resolution are needed.